V-ATPases are active transporters necessary for cellular and systemic pH homeostasis. Inhibition of V- ATPase function disturbs cellular pH;and because the pH influences nearly all cellular processes, most cells die without functional V-ATPases. Inhibition of V-ATPase pumps prevents virulence by Candida and other pathogens because trafficking of virulence-related proteins rely on V-ATPase-dependent sorting mechanisms. This study is aimed at identifying small molecules that inhibit yeast V-ATPases with high potency in vivo. V- ATPase inhibitors with anti-fungal activity are expected to block virulence. We propose high-throughput screening (HTS) of the Molecular Libraries Probe Centers Network (MLPCN) library of small molecules (~330,000 compounds). To accomplish this aim we developed two HTS flow cytometry assays in collaboration with the University of New Mexico Center for Molecular Discovery. These assays use pH-dependent fluorescence probes and HyperCyt@ to monitor pH changes in the cytosol (Primary Assays) and vacuolar lumen (Secondary Assays) of the model yeast S. cerevisiae. For the Primary Assays the pH-sensitive GFP (pHLuorin) trapped in the cytosol is used to monitor cytosolic acidification. Since acidification of the cytosol by inhibition of V-ATPase pumps goes together with alkalinization of the vacuolar lumen, compounds that lower the cytosolic pH will be questioned in Secondary Assays to assess their effect on the vacuolar pH. For the Secondary Assays the compounds are added to cells stained with BCECF trapped in the vacuoles. V-ATPase inhibitors will increase the vacuolar pH and enhance the fluorescence intensity of BCECF. Inhibitors will be confirmed in medium-throughput Tertiary Assays that measure ATP hydrolysis directly in vacuolar membranes. Anti-fungal selectivity will be enhanced in the Tertiary Assays by comparing wild-type membranes, which have normal V-ATPase function, and mutant membranes, which have V-ATPases resistant to bafilomycin and concanamycin. The inhibitors that mimic concanamycin and bafilomycin (inhibit both yeast and mammalian V-ATPases) and the inhibitors that act by other mechanisms will be identified. Further studies will establish the effects that inhibitors have on the enzyme kinetics and assembly. Small molecules identified in this study will offer opportunities to develop new therapeutic tools against pathogens such as Candida and hundreds of emerging opportunistic fungal infections. V-ATPase inhibitors will also contribute knowledge to the understanding of the molecular mechanisms governing pH regulation and fungal diseases. PUBLIC HEALTH RELEVANCE: This study proposes high throughput screening of the MLPCN library to identify small molecules that inhibit fungal V-ATPase pumps with high potency in vivo. V-ATPase pumps are proteins necessary for fugal virulence and yeast V-ATPase inhibitors are expected to block virulence. V-ATPase inhibitors identified in this study will offer opportunities to develop new therapeutic tools to treat Candida and hundreds of others emerging opportunistic fungal infections. They will also contribute knowledge to the understanding of the molecular mechanisms governing fungal diseases.